The isolation of commercially useful quantities of polypeptides from their natural biological sources is often difficult and expensive. This difficulty is usually due to either a scarcity of source material or the presence of small amounts of polypeptide per unit of source material. Commonly, both of these factors operate simultaneously.
Advances in biotechnology have eased the production and isolation of polypeptides and proteins. A DNA sequence coding for a desired polypeptide or protein may be inserted into a cloning vector along with appropriate regulatory sequences. The introduction of such a vector into a compatible host cell will result in production by the host cell of the polypeptide encoded by the inserted DNA sequence. Because of the cost of culturing cells and isolating the desired proteins from the cultures, the biotechnology industry has long sought methods to increase the yield of protein product produced per unit volume of transformed cell culture as well as the yield per unit time.
The level of production of a product by recombinant host cells is controlled by three major factors: (i) the number of copies of a DNA sequence coding for the product ("copy number") in the cell; (ii) the efficiency with which this DNA sequence is transcribed into messenger RNA; and (iii) the efficiency with which this messenger RNA is translated to generate the protein product. Efficiency of transcription and translation (which together comprise expression) is in turn dependent upon regulatory sequences located upstream and downstream of, and in some instances internal to, the product gene.
None of the methods known in the art for introducing foreign DNA into eukaryotic cells results in stable integration of greater than approximately twenty copies of this foreign DNA into the host cell genome. Instead, these low copy number integrants must be treated to amplify their integrated foreign DNA, in order to produce high copy number integrants. The most widely used procedure to obtain high copy number integrants utilizes the dihydrofolate reductase ("DHFR") gene.
Mammalian cells which contain multiple copies of the DHFR gene are selected when a culture of these cells is subjected to sequentially increasing concentrations of methotrexate (Alt et al., "Selective Multiplication of Dihydrofolate Reductase Genes in Methotrexate-Resistant Variants of Cultured Murine Cells", J. Biol. Chem., 253, pp. 1357-79 (1978)). DHFR is an essential enzyme for cell survival. Since methotrexate ("MTX") is a competitive inhibitor of DHRF, only those cells that have increased their DHFR content (e.g. by amplifying the resident DHRF gene) to offset MTX inhibition will survive. Furthermore, as the MTX concentration is sequentially increased, cells will require increasing amounts of DHFR, and thus higher DHFR gene copy numbers, to survive. This is the basis of the DHFR gene amplification procedure.
One indication that the DHFR gene might be useful in the amplification of the cotransfected genes was the report that when Escherichia coli plasmid pBR322 was cotransfected (introduced together) with genomic DNA containing a MTX-resistant DHFR gene into mouse cells, the pBR322 DNA was also amplified by MTX selection (Wigler et al., "Transformation of Mammalian Cells with an Amplifiable Dominant-Acting Gene", Proc. Natl. Acad. Sci. USA, 77(6), pp. 3567-70 (1980)). However, most of the Wigler transfectants did not amplify the pBR322 DNA more than several-fold.
The generation of very high copy number integrants was made possible by the isolation of Chinese hamster cells deficient in native DHFR activity ("DHFR.sup.- CHO cells") (Urlaub and Chasin, "Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity", Proc. Natl. Acad. Sci. USA, 77(7), 4216-20 (1980). Transfection of these DHFR.sup.- CHO cells with a plasmid containing both the DHFR gene and the E. coli gpt gene, followed by MTX selection, produced recombinant host cells which had amplified the gpt gene approximately 50-fold (Ringold et al., "Co-expression and Amplification of Dihydrofolate Reductase cDNA and the Escherichia coli XGPRT gene in Chinese Hamster Ovary Cells", J. Mol. Appl. Genet., 1, pp. 165-75 (1981)).
In a more dramatic example of the possibility for amplification of non-selectable genes using this technique, transfection of DHFR.sup.- CHO cells with plasmids containing both the murine DHFR gene and the SV40 early region, followed by sequential step-wise increases in the MTX concentration of the growth medium, produced cells containing up to 1000 copies of the transforming DNA (Kaufman and Sharp, "Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol., 159, pp. 601-21 (1982)).
While the DHFR/MTX amplification procedure produces cells with amplified copies of transfected DNA, it has several serious drawbacks. These drawbacks include the slowness of the procedure, the necessity of using DHFR.sup.- cells to obtain significant amplification, and the fluidity of amplified DNA.
To select recombinant host cells which have amplified transfected DNA to a very high copy number, they must be subjected to sequential step-wise increases in the MTX concentration of the growth medium. This is a lengthy process. In our hands, six to ten months are required to achieve a several hundred-fold amplification. Obviously, a more expeditious procedure would be desirable.
Another drawback of the DHFR/MTX amplification procedure is that it does not work well for cells that contain a DHFR gene ("DHFR.sup.+ cells"). At best, only a fifty-fold amplification of transfected DNA has been reported in DHFR.sup.+ cells (Wigler et al., "Transformation of Mammalian Cells with an Amplifiable Dominant-Acting Gene", Proc. Natl. Acad. Sci. USA, 77(6), pp. 3567-70 (1980)). The production of DHFR.sup.- cells from DHFR.sup.+ cells, if possible at all for a given cell type, is lengthy and laborious (Urlaub and Chasin, "Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity", Proc. Natl. Acad. Sci. USA, 77(7), pp. 4216-20 (1980)). Since all mammalian cells possess the DHFR gene, a worker looking for significant amplification of transfected DNA would be restricted to using DHFR.sup.- CHO cells unless he was willing to face the ordeal of creating a new DHFR.sup.- cell type.
An additional drawback of DHFR/MTX amplification is that not all sequences contained within transfected DNA will be amplified to the same degree (Kaufman and Sharp, "Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene", J. Mol. Biol., 159, pp. 601-21 (19B2)). Of equal concern are reports of deletions and rearrangements within amplified DNA (see, e.g., Kaufman and Sharp, id.; Schimke, "Gene Amplification in Cultured Animal Cells", Cell, 37, pp. 705-13 (1984)).